Serological evidence of infection with Toxoplasma gondii can be seen in up to 40% of humans. Infection with this Apicomplexan parasite can cause a severe congenital infection with an associated fetopathy. T. gondii is also a cause of opportunistic infections in immune compromised hosts. The predilection of this parasite for the central nervous system (CNS) causing necrotizing encephalitis and the eye causing chorioretinitis constitutes its major threat to patients. In both congenital infection and in immune compromised patients CNS and ocular toxoplasmosis is believed to be a consequence of the reactivation of chronic or latent infection due to persistent tissue cysts (bradyzoites) of T. gondii. The presence of these tissues cysts in brain and muscle are critical for persistence of this protozoan parasite. We have established that cysts are enclosed in highly glycosylated cyst wall, but the role(s) of glycosylation in persistence and the pathogenesis of parasites are not fully understood. Several glycoproteins are known to be expressed and localized to the cyst wall. Our laboratory group has demonstrated that one of these cyst wall glycoproteins, CST1, is critical in the differentiation of the parasites and formation of the cyst wall structure. Furthermore, we have demonstrated that CST1 is primarily modified by o-linked glycosylation and that this glycosylation of CST1 is vital for stability of the cyst wall. This R21 will focus on the development of a novel approach and reagents that we have developed that employ click chemistry and porcine leukocyte esterase (PLE) to allow selective labeling of glycoproteins in this obligate intracellular organism. Using genetically modified T. gondii strains that have various glycosyltransferases (ppGalNAc-Ts) knockouts we will characterize the o-glycoproteome of T. gondii during cyst formation. The proposed studies utilize a proteomic approach employing click chemistry and mass spectrometry to validate our new PLE click chemistry strategy. This PLE technique is applicable to many compounds and, once validated, will provide a tool for selective delivery of small molecules to obligate intracellular pathogens, permitting selective labeling of these organisms as well as selective targeting of pathways. The interdisciplinary team that has been assembled for this project includes internationally recognized expertise in T. gondii bradyzoite biology (Dr. Louis Weiss), orthogonal click chemistry (Dr. Peng Wu) and glycobiology (Dr. Pamela Stanley).